Blood Oxidative Stress Modulates Alveolar Bone Loss in Chronically Stressed Rats

Abstract

We aimed to investigate the effects of chronic stress (CS) on experimental periodontitis (EP) in rats. For this, 28 Wistar rats were divided into four groups: control, ligature-induced experimental periodontitis (EP), chronic stress (CS; by physical restraint model) and CS+EP (association of chronic stress and ligature-induced periodontitis). The experimental period lasted 30 days, including exposure to CS every day and ligature was performed on the 15th experimental day. After 30 days, the animals were submitted to the behavioral test of the elevated plus maze (EPM). Next, rats were euthanized for blood and mandible collection in order to evaluate the oxidative biochemistry (by nitric oxide (NO), reduced-glutathione activity (GSH), and thiobarbituric acid reactive substance levels (TBARS)) and alveolar bone characterization (by morphometric, micro-CT, and immunohistochemistry), respectively. The behavioral parameters evaluated in EPM indicated higher anxiogenic activity in the CS and CS+EP, groups, which is a behavioral reflex of CS. The results showed that CS was able to change the blood oxidative biochemistry in CS and CS+EP groups, decrease GSH activity in the blood, and increase the NO and TBARS concentrations. Thus, CS induces oxidative blood imbalance, which can potentialize or generate morphological, structural, and metabolic damages to the alveolar bone.

Keywords: alveolar bone loss; chronic stress; experimental periodontitis; ligatures.

Figure 1

Effects of experimentally induced chronic immobilization stress and ligature-induced periodontitis on anxiety-like behavior of male Wistar rats (90-days-old, n = 28). (A) Entrance frequency in open arms (%) and (B) time in the open arms (%) in the elevate plus maze; results are expressed as mean ± standard error. One-way ANOVA and Tukey’s post-hoc test, p < 0.05. Similar overwritten letters did not show statistically significant differences. EP: ligature-induced experimental periodontitis group; CS: chronic stress by physical restraint model; CS+EP: association of chronic stress and ligature-induced periodontitis.

Figure 2

Effects of experimentally induced chronic immobilization stress and ligature-induced periodontitis on oxidative biochemical paraments of male Wistar rats (90-days-old, n = 28) in the blood. Results are expressed as mean ± standard error of mean of control percentage of (A) glutathione (GSH) levels; (B) nitric oxide (NO) levels; and (C) lipid peroxidation (TBARS). One-way ANOVA and Tukey’s post-hoc test, p < 0.05. Similar overwritten letters do not reveal statistically significant differences. EP: ligature-induced experimental periodontitis group; CS: chronic stress by physical restraint model; CS+EP: association of chronic stress and ligature-induced periodontitis.

Figure 3

Effects of experimentally induced chronic immobilization stress and ligature-induced periodontitis on RANK and RANK-L expression of the alveolar bone (blue arrows) of male Wistar rats (90-days-old, n = 28). Representative photomicrographs of the control group; experimental periodontitis group (EP); chronic stress group (CS); and chronic stress and experimental periodontitis group (CS+EP). Results are expressed as mean ± standard error of immunohistochemistry score (IHC score) of RANK and RANKL expression. Kruskal–Wallis test, p < 0.05. Similar overwritten letters did not reveal statistically significant differences. EP: ligature-induced experimental periodontitis group; CS: chronic stress by physical restraint model; CS+EP: association of chronic stress and ligature-induced periodontitis. Scale bar: 100 µm. Cementum (★) and periodontal ligament (*).

Figure 4

Effects of experimentally induced chronic immobilization stress and ligature-induced periodontitis on the quality of the alveolar bone of male Wistar rats (90-days-old, n = 28). Representative three-dimensional images of hemimandibles of the control group; experimental periodontitis group (EP); chronic stress group (CS); and chronic stress and periodontitis group (CS+EP). The representative image of the interradicular region, close to the furcation area, chosen to the standardized region of interest (ROI) for the analyses. Results are expressed as mean ± standard error for micro-CT parameters: (A) distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) in millimeters (mm); (B) trabecular thickness (Tb.Th; mm); (C) trabecular spacing (Tb.Sp; mm); (D) mean number of trabecular per unit length (Tb.N); and (E) the percentage of bone volume in relation to the total measured area (BV/TV; %). One-way ANOVA and Tukey’s post-hoc test, p < 0.05. Similar overwritten letters did not reveal statistically significant differences. Scale bar: 1 mm.

Figure 5

Effects of experimentally induced chronic immobilization stress and ligature-induced periodontitis on the alveolar bone of male Wistar rats (90-days-old, n = 28). Representative photomicrographs of hemimandibles of the (A) control group; (B) experimental periodontitis group (EP); (C) chronic stress group (CS), and (D) chronic stress and experimental periodontitis group (CS+EP). Results are expressed as mean ± standard error of the mean of (E) alveolar bone-loss area (highlighted in yellow). One-way ANOVA and Tukey’s post-hoc test, p < 0.05. Similar overwritten letters do not show statistically significant differences. EP: ligature-induced experimental periodontitis group; CS: chronic stress by physical restraint model; CS+EP: association of chronic stress and ligature-induced periodontitis. Scale bar: 0.5 mm.

Figure 6

Methodological flow: (1) sample description and experimental steps; (2) elevated plus maze is the behavioral test adopted to investigate indirect parameters of chronic stress; (3) morphometric, microtomographic and immunohistochemical morphological analyzes in the hemi-mandibular bone. (4) Investigation of oxidative biochemistry determination of glutathione levels (GSH), lipid peroxidation (TBARS), nitric oxide (NO) levels.